U.S. patent application number 12/462189 was filed with the patent office on 2010-06-03 for method for making monoclonal antibodies and cross-reactive antibodies obtainable by the method.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Avi J. Ashkenazi, Anan Chuntharapai, K. Jin Kim.
Application Number | 20100136624 12/462189 |
Document ID | / |
Family ID | 26780401 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100136624 |
Kind Code |
A1 |
Ashkenazi; Avi J. ; et
al. |
June 3, 2010 |
Method for making monoclonal antibodies and cross-reactive
antibodies obtainable by the method
Abstract
A method of making monoclonal antibodies according to a mixed
antigen immunization protocol is described. In addition, antibodies
obtainable by the method are disclosed which specifically
cross-react with two or more different receptors to which Apo-2
ligand (Apo-2L) can bind.
Inventors: |
Ashkenazi; Avi J.; (San
Mateo, CA) ; Chuntharapai; Anan; (Colma, CA) ;
Kim; K. Jin; (Los Altos, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
26780401 |
Appl. No.: |
12/462189 |
Filed: |
July 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11789417 |
Apr 24, 2007 |
7592439 |
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12462189 |
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11455062 |
Jun 15, 2006 |
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11789417 |
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11104779 |
Apr 13, 2005 |
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11455062 |
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09828739 |
Apr 9, 2001 |
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11104779 |
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09329633 |
Jun 10, 1999 |
6252050 |
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09828739 |
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60089253 |
Jun 12, 1998 |
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Current U.S.
Class: |
435/69.6 ;
435/320.1; 435/372; 536/23.53 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 16/2875 20130101; C07K 14/70575 20130101; A61K 2039/505
20130101; C07K 2317/73 20130101; C07K 16/2878 20130101; C07K
2319/30 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/69.6 ;
536/23.53; 435/320.1; 435/372 |
International
Class: |
C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C12P 21/00 20060101 C12P021/00 |
Claims
1. An isolated nucleic acid comprising DNA encoding an antibody
which specifically cross-reacts with two or more different Apo-2L
receptors.
2. The nucleic acid of claim 1 wherein the antibody comprises a
monoclonal antibody.
3. The nucleic acid of claim 1 wherein the antibody specifically
binds to Apo-2 polypeptide and further specifically cross-reacts
with another Apo-2L receptor.
4. The nucleic acid of claim 1 wherein the antibody specifically
binds to Apo-2 polypeptide and further specifically cross-reacts
with DR4.
5. The nucleic acid of claim 1 wherein the antibody is an agonistic
antibody.
6. The nucleic acid of claim 1 wherein the antibody is a blocking
antibody.
7. The nucleic acid of claim 1 wherein the antibody is an antibody
fragment.
8. The nucleic acid of claim 1 wherein the antibody comprises
non-human hypervariable region residues and human framework region
residues.
9. The nucleic acid of claim 1 wherein the antibody is a human
antibody.
10. The nucleic acid of claim 1 wherein the Apo-2L receptors are
native sequence Apo-2L receptors.
11. The nucleic acid of claim 5 wherein the agonistic antibody
binds to Apo-2 polypeptide or DR4.
12. An isolated nucleic acid comprising DNA encoding an antibody
having the biological characteristics of a monoclonal antibody
selected from the group consisting of 3H1.18.10 (produced by the
hybridoma having ATCC Accession No. HB-12535), 3H3.14.5 (produced
by the hybridoma having ATCC Accession No. HB-12534) and 3D5.1.10
(produced by the hybridoma having ATCC Accession No. HB-12536).
13. The nucleic acid of claim 12 wherein the antibody binds to the
same epitope as the epitope to which a monoclonal antibody selected
from the group consisting of 3H1.18.10 (produced by the hybridoma
having ATCC Accession No. HB-12535), 3H3.14.5 (produced by the
hybridoma having ATCC Accession No. HB-12534) and 3D5.1.10
(produced by the hybridoma having ATCC Accession No. HB-12536)
binds.
14. The nucleic acid of claim 12 wherein the antibody has the
hypervariable region residues of a monoclonal antibody selected
from the group consisting of 3H1.18.10 (produced by the hybridoma
having ATCC Accession No. HB-12535), 3H3.14.5 (produced by the
hybridoma having ATCC Accession No. HB-12534) and 3D5.1.10
(produced by the hybridoma having ATCC Accession No. HB-12536).
15. A vector comprising the nucleic acid of claim 1.
16. A host cell comprising the nucleic acid of claim 1.
17. A method of producing an antibody comprising culturing the host
cell of claim 16 under conditions wherein the DNA is expressed.
18. The method of claim 17 further comprising recovering the
antibody from the host cell culture.
19. The method of claim 18 further comprising combining the
recovered antibody with a pharmaceutically acceptable carrier.
20. The method of claim 18 further comprising conjugating the
recovered antibody with a heterologous molecule.
21. The method of claim 20 wherein the heterologous molecule is
polyethylene glycol, a label or a cytotoxic agent.
Description
RELATED APPLICATION
[0001] This is a divisional of non-provisional application Ser. No.
09/329,633 filed Jun. 10, 1999 which claims priority under 35 USC
119(e) to provisional application No. 60/089,253 filed 12 Jun.
1998, the contents of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to a method for making
monoclonal antibodies. The invention further pertains to antibodies
obtainable by the method which specifically cross-react with two or
more different receptors to which Apo-2 ligand (Apo-2L) can
bind.
[0004] 2. Description of Related Art
[0005] Native antibodies are synthesized primarily by specialized
lymphocytes called "plasma cells". Production of a strong antibody
response in a host animal is controlled by inducing and regulating
the differentiation of B cells into these plasma cells. This
differentiation involves virgin B cells (which have a modified
antibody as a cell-surface antigen receptor and do not secrete
antibodies) becoming activated B cells (which both secrete
antibodies and have cell-surface antibodies), then plasma cells
(which are highly specialized antibody factories with no surface
antigen receptors). This differentiation process is influenced by
the presence of antigen and by cellular communication between B
cells and helper T cells.
[0006] Because of their ability to bind selectively to an antigen
of interest, antibodies have been used widely for research,
diagnostic and therapeutic applications. The potential uses for
antibodies were expanded with the development of monoclonal
antibodies. In contrast to polyclonal antiserum, which includes a
mixture of antibodies directed against different epitopes,
monoclonal antibodies are directed against a single determinant or
epitope on the antigen and are homogeneous. Moreover, monoclonal
antibodies can be produced in unlimited quantities.
[0007] The seminal work by Kohler and Milstein described the first
method for obtaining hybridomas that can produce monoclonal
antibodies [Kohler and Milstein Nature 256:495 (1975)]. In this
method, an antibody-secreting immune cell, isolated from an
immunized mouse, is fused with a myeloma cell, a type of B cell
tumor. The resultant hybrid cells (i.e. hybridomas) can be
maintained in vitro and continue to secrete antibodies with a
defined specificity.
[0008] Since murine monoclonal antibodies are derived from mice,
their use as therapeutic agents in humans is limited because of the
human anti-mouse response that occurs upon administration of the
murine antibody to a patient. Accordingly, researchers have
engineered non-human antibodies to make them appear more human.
Such engineered antibodies are called "chimeric" antibodies; in
which a non-human antigen-binding domain is coupled to a human
constant domain (Cabilly et al., U.S. Pat. No. 4,816,567). The
isotype of the human constant domain may be selected to tailor the
chimeric antibody for participation in antibody-dependent cellular
cytotoxicity (ADCC) and complement-dependent cytotoxicity. In a
further effort to resolve the antigen binding functions of
antibodies and to minimize the use of heterologous sequences in
human antibodies, Winter and colleagues [(Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);
Verhoeyen et al., Science 239:1534-1536 (1988)] have substituted
rodent complementarity determining region (CDR) residues for the
corresponding segments of a human antibody to generate humanized
antibodies. As used herein, the term "humanized" antibody is an
embodiment of chimeric antibodies wherein substantially less than
an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which CDR
residues and possibly some framework region (FR) residues are
substituted by residues from analogous sites in rodent
antibodies.
[0009] Other groups have developed methods for making fully "human"
monoclonal antibodies. Such antibodies may be generated by
immortalizing a human cell secreting a specific antibody using an
Epstein-Barr virus (EBV) [Steinitz et al. Nature 269:420-422
(1977)]; or by preparing a human-human hybridoma secreting the
monoclonal antibody [Olsson et al. PNAS (USA) 77:5429-5431 (1980)].
Human antibodies can also be derived from phage-display libraries
[Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J.
Mol. Biol., 222:581-597 (1992); Vaughan et al. Nature Biotech
14:309 (1996)].
[0010] Alternatively, human antibodies have been made in transgenic
laboratory animals, in which human immunoglobulin loci have been
introduced into the animal and the endogenous immunoglobulin genes
are partially or completely inactivated [Fishwild et al. Nature
Biotech. 14:845-851 (1996); and Mendez et al. Nature Genetics
15:146-156 (1997)].
SUMMARY OF THE INVENTION
[0011] The present invention, in one aspect, provides a method for
making monoclonal antibodies wherein an animal is immunized with
two or more different antigens and monoclonal antibodies are made
and identified which bind to each antigen. Surprisingly, it was
discovered herein that sera titers from animals immunized with a
mixture of different antigens were similar to those achieved in
animals immunized with a single antigen.
[0012] This method is thought to be useful for reducing the number
of animals that need to be immunized and sacrificed in order to
make two or more monoclonal antibodies with differing
antigen-binding specificities.
[0013] Moreover, it was discovered that the method was useful for
making antibodies that cross-reacted with two or more different
antigens. For example, antibodies were made which specifically
cross-reacted with two or more different Apo-2L receptors.
[0014] Accordingly, the invention provides a method for making
antibodies comprising the following steps:
[0015] (a) immunizing an animal with two or more different antigens
so as to generate polyclonal antibodies against each antigen in the
animal;
[0016] (b) preparing monoclonal antibodies using immune cells of
the immunized animal which produce said polyclonal antibodies;
and
[0017] (c) screening said monoclonal antibodies to identify one or
more monoclonal antibodies that bind to each antigen. In the
screening step, one finds at least one monoclonal antibody against
at least two different antigens. Preferably, at least one
monoclonal antibody is found for each antigen with which the animal
was immunized.
[0018] Preferably, the animal is immunized with a composition
comprising a mixture of the two or more different antigens; and
step (b) comprises fusing immune cells from the immunized animal
with myeloma cells in order to generate hybridoma cell lines
producing the monoclonal antibodies.
[0019] In one embodiment, the method further comprises identifying
one or more monoclonal antibodies that cross-react with two or more
of the different antigens.
[0020] The invention further provides a monoclonal antibody that
has been made according to the above method (e.g. one that
cross-reacts with two or more structurally or functionally related
antigens).
[0021] The invention also relates to an antibody that specifically
cross-reacts with two or more different Apo-2L receptors; e.g.
which specifically binds to Apo-2 polypeptide and further
specifically cross-reacts with another Apo-2L receptor.
[0022] The present application further supplies a monoclonal
antibody which has the biological characteristics of a monoclonal
antibody selected from the group consisting of 3H1.18.10, 3H3.14.5
and 3D5.1.10.
[0023] Moreover, the invention provides hybridoma cell lines that
produce any of the monoclonal antibodies disclosed herein.
[0024] The invention also relates to isolated nucleic acid
comprising DNA encoding an antibody as herein disclosed; a vector
comprising the nucleic acid; a host cell comprising the vector; a
method of producing an antibody comprising culturing the host cell
under conditions wherein the DNA is expressed and, optionally,
further comprising recovering the antibody from the host cell
culture.
[0025] The invention further provides a composition comprising an
antibody as described herein and a carrier.
[0026] In addition, a method of inducing apoptosis in mammalian
cancer cells is provided which comprises exposing mammalian cancer
cells to an effective amount of a cross-reactive, agonistic
anti-Apo-2L receptor antibody as disclosed herein.
[0027] The invention further pertains to an article of manufacture
comprising a container and a composition contained within said
container, wherein the composition includes an antibody as
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 depicts an exemplary mixed antigen immunization
scheme for the immunogens: Apo-2-IgG, DR4-IgG, DcR1-IgG and
DcR2-IgG.
[0029] FIG. 2 illustrates single antigen immunization schemes for
the antigens: Apo-2-IgG, DR4-IgG and DcR2-IgG.
[0030] FIG. 3 shows antigen specific sera titers of mice immunized
with DR4-IgG, Apo-2-IgG or DcR2-IgG individually. Sera were
collected from Balb/c mice (5 mice/group) which were immunized 10
times into Foot Pad (F.P.) with 1 .mu.g of each immunoadhesin
molecule in MPL-TDM. The activity toward human IgG Fc portion was
preadsorbed by incubating 100 ml of sera (1:500 dilution in PBS)
with 3 mg per 50 ml of CD4-IgG for 1 hr at room temperature (RT).
Serial dilutions of this preadsorbed sera were than prepared in
PBS. The antigen specific activities of this preadsorbed sera were
determined in a capture ELISA using the specific antigen coated
microtiter wells.
[0031] FIG. 4 shows antigen specific sera titers of mice immunized
with DR4-IgG, Apo-2-IgG, DcR1-IgG and DcR2-IgG together. Mice were
immunized into F.P. with a mixture of DR4-IgG, Apo-2-IgG, DcR1-IgG
and DcR2-IgG (mice were immunized 14 times; DcR2-IgG was only
included in the mixture for the final 6 immunizations). 1 .mu.g per
injection of each immunogen was used. The activity to human IgG Fc
in the sera was adsorbed by incubating with CD4-IgG as described
above. The activity of this preadsorbed sera specific for each
antigen was determined in a capture ELISA using the microtiter
wells coated with the specific antigen.
[0032] FIGS. 5A and 5B show the nucleotide sequence of a native
sequence human Apo-2 cDNA (SEQ ID NO:1) and its derived amino acid
sequence (SEQ ID NO:2).
[0033] FIGS. 6A, 6B and 6C depict antibody binding to Apo2-L
receptors: DR4, Apo-2, DcR1 and DcR2 as determined by ELISA. The
antibodies are: 3H3.14.5 (FIG. 6A), 3H1.18.10 (FIG. 6B), and
3D5.1.10 (FIG. 6C).
[0034] FIGS. 7A, 7B and 7C show FACS analysis for antibodies
3H1.18.10 (FIG. 7A), 3H3.14.5 (FIG. 7B), and 3D5.1.10 (FIG. 7C)
[illustrated by bold lines] as compared to IgG controls [dotted
lines]. The antibodies all recognized Apo-2 expressed in human 9D
cells.
[0035] FIG. 8 depicts apoptosis induced by antibodies 3H1.18.10
(3H1), 3H3.14.5 (3H3) and 3D5.1.10 (3D5), an isotype-matched
control (IgG), and Apo-2L.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0036] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including agonist,
antagonist, and blocking or neutralizing antibodies) and antibody
compositions with polyepitopic specificity.
[0037] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally-occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen.
[0038] The monoclonal antibodies herein include hybrid and
recombinant antibodies produced by splicing a variable (including
hypervariable) domain of an antibody with a constant domain, or a
light chain with a heavy chain, or a chain from one species with a
chain from another species, or fusions with heterologous proteins,
regardless of species of origin or immunoglobulin class or subclass
designation, as well as antibody fragments (e.g., Fab, Fab',
F(ab').sub.2, and Fv), so long as they exhibit the desired
biological activity. See, e.g. U.S. Pat. No. 4,816,567 and Mage et
al., in Monoclonal Antibody Production Techniques and Applications,
pp. 79-97 (Marcel Dekker, Inc.: New York, 1987).
[0039] Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies of the present invention may be made by
the hybridoma method first described by Kohler and Milstein,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
such as described in U.S. Pat. No. 4,816,567. The "monoclonal
antibodies" may also be isolated from phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990), for example.
[0040] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the scFv to form the
desired structure for antigen binding. For a review of scFv see,
e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol.
113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.
269-315 (1994).
[0041] "Humanized" forms of non-human (e.g. murine) antibodies are
specific chimeric immunoglobulins, immunoglobulin chains, or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a hypervariable region of the recipient are
replaced by residues from a hypervariable region of a non-human
species (donor antibody) such as mouse, rat, or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues which are found neither in
the recipient antibody or the donor antibody. These modifications
are made to further refine and optimize antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
FR regions are those of a human immunoglobulin consensus sequence.
The humanized antibody optimally also will comprise at least a
portion of an immunoglobulin constant region or domain (Fc),
typically that of a human immunoglobulin.
[0042] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (i.e.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (i.e. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0043] The terms "Apo-2 ligand" or "Apo-2L" refer to the Apo-2L
polypeptides disclosed in WO97/25428, published 17 Jul. 1997 and
expressly incorporated herein by reference. For purposes of the
present application, these terms also refer to the polypeptides
disclosed in WO97/01633, published 16 January, 1997 and expressly
incorporated herein by reference.
[0044] An "Apo-2L receptor" is a polypeptide to which Apo-2L (as
herein defined) can specifically bind. The term "Apo-2L receptor"
when used herein encompasses native sequence Apo-2L receptors and
variants thereof (which are further defined herein). These terms
encompass Apo-2L receptor from a variety of mammals, including
humans. The Apo-2L receptor may be isolated from a variety of
sources, such as from human tissue types or from another source, or
prepared by recombinant or synthetic methods. Examples of "native
sequence" Apo-2L receptors include Apo-2 polypeptide (as described
herein below), native sequence "DR4" as described in Pan et al.
Science 276:111-113 (1997); native sequence "decoy receptor 1" or
"DcR1" as in Sheridan et al., Science 277:818-821 (1997); and
native sequence "decoy receptor 2" or "DcR2" as in Marsters et al.
Curr. Biol. 7:1003-1006 (1997) and native sequence osteoprotegerin
[see Simonet et al. Cell 89:309-319 (1997) and Emery et al. J.
Interferon and Cytokine Research 18(5): A47 Abstract 2.17
(1998)]
[0045] The terms "Apo-2 polypeptide" and "Apo-2" when used herein
encompass native sequence Apo-2 and Apo-2 variants (which are
further defined herein). These terms encompass Apo-2 from a variety
of mammals, including humans. The Apo-2 may be isolated from a
variety of sources, such as from human tissue types or from another
source, or prepared by recombinant or synthetic methods.
[0046] A "native sequence" polypeptide (e.g. "native sequence
Apo-2") comprises a polypeptide having the same amino acid sequence
as a polypeptide derived from nature. Thus, a native sequence
polypeptide can have the amino acid sequence of naturally-occurring
polypeptide from any mammal. Such native sequence polypeptide can
be isolated from nature or can be produced by recombinant or
synthetic means. The term "native sequence" polypeptide
specifically encompasses naturally-occurring truncated or secreted
forms of the polypeptide (e.g., an extracellular domain sequence),
naturally-occurring variant forms (e.g., alternatively spliced
forms) and naturally-occurring allelic variants of the
polypeptide.
[0047] A naturally-occurring variant form of Apo-2 includes an
Apo-2 having an amino acid substitution at residue 410 in the amino
acid sequence shown in FIG. 5 (SEQ ID NO:2). In one embodiment of
such naturally-occurring variant form, the leucine residue at
position 410 is substituted by a methionine residue. In FIG. 5 (SEQ
ID NO:2), the amino acid residue at position 410 is identified as
"Xaa" to indicate that the amino acid may, optionally, be either
leucine or methionine. In FIG. 10 (SEQ ID NO:2), the nucleotide at
position 1367 is identified as "W" to indicate that the nucleotide
may be either adenine (A) or thymine (T) or uracil (U). In one
embodiment of the invention, the native sequence Apo-2 is a mature
or full-length native sequence Apo-2 comprising amino acids 1 to
411 of FIG. 5 (SEQ ID NO:2). Optionally, the Apo-2 is obtained or
obtainable by expressing the polypeptide encoded by the cDNA insert
of the vector deposited as ATCC 209021.
[0048] An "extracellular domain" or "ECD" (e.g. "Apo-2
extracellular domain" or "Apo-2 ECD") refers to a form of a
receptor polypeptide which is essentially free of the transmembrane
and cytoplasmic domains of the receptor. Ordinarily, the ECD will
have less than 1% of such transmembrane and/or cytoplasmic domains
and preferably, will have less than 0.5% of such domains.
Optionally, Apo-2 ECD will comprise amino acid residues 54 to 182
of FIG. 5 (SEQ ID NO:2) or amino acid residues 1 to 182 of FIG. 5
(SEQ ID NO:2). Optionally, Apo-2 ECD will comprise one or more
cysteine-rich domains, and preferably, one or both of the
cysteine-rich domains identified for the sequence shown in Sheridan
et al., Science 277:818-821 (1997). It will be understood by the
skilled artisan that the transmembrane domain identified for the
Apo-2 polypeptide herein is identified pursuant to criteria
routinely employed in the art for identifying that type of
hydrophobic domain. The exact boundaries of a transmembrane domain
may vary but most likely by no more than about 5 amino acids at
either end of the domain specifically mentioned herein.
[0049] A polypeptide "variant" (e.g. "Apo-2 variant") means a
biologically active polypeptide having at least about 80% amino
acid sequence identity with the native sequence polypeptide, e.g.
with Apo-2 having the deduced amino acid sequence shown in FIG. 5
(SEQ ID NO:2) for a full-length native sequence human Apo-2 or the
sequences identified herein for Apo-2 ECD or death domain. Such
variants include, for instance, polypeptides wherein one or more
amino acid residues are added, or deleted, at the N- or C-terminus
of the polypeptide [e.g. in the case of Apo-2 in the sequence of
FIG. 5 (SEQ ID NO:2) or the sequences identified herein for Apo-2
ECD or death domain].
[0050] Examples of "antibody variants" include humanized variants
of non-human antibodies, "affinity matured" antibodies (see, e.g.
Hawkins et al. J. Mol. Biol. 254: 889-896 [1992] and Lowman et al.
Biochemistry 30(45): 10832-10837 [1991]) and antibody mutants with
altered effector function(s) (see, e.g., U.S. Pat. No. 5,648,260
issued on Jul. 15, 1997, expressly incorporated herein by
reference).
[0051] Ordinarily, a variant will have at least about 80% amino
acid sequence identity, more preferably at least about 90% amino
acid sequence identity, and even more preferably at least about 95%
amino acid sequence identity with native sequence [e.g. for Apo-2,
with the amino acid sequence of FIG. 5 (SEQ ID NO:2) or the
sequences identified herein for Apo-2 ECD or death domain].
[0052] "Percent (%) amino acid sequence identity" is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in the native sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as ALIGN.TM. or Megalign (DNASTAR) software. Those
skilled in the art can determine appropriate parameters for
measuring alignment, including any algorithms needed to achieve
maximal alignment over the full length of the sequences being
compared.
[0053] The term "epitope tagged" when used herein refers to a
polypeptide, or a domain sequence thereof, fused to a "tag
polypeptide". The tag polypeptide has enough residues to provide an
epitope against which an antibody can be made, yet is short enough
such that it does not interfere with activity of the polypeptide.
The tag polypeptide preferably also is fairly unique so that the
antibody does not substantially cross-react with other epitopes.
Suitable tag polypeptides generally have at least six amino acid
residues and usually between about 8 to about 50 amino acid
residues (preferably, between about 10 to about 20 residues).
[0054] "Biologically active" and "desired biological activity" with
respect to an Apo-2L receptor for the purposes herein means (1)
having the ability to modulate apoptosis (either in an agonistic or
stimulating manner or in an antagonistic or blocking manner) in at
least one type of mammalian cell in vivo or ex vivo; (2) having the
ability to bind Apo-2 ligand; or (3) having the ability to modulate
Apo-2 ligand signaling and Apo-2 ligand activity.
[0055] The terms "apoptosis" and "apoptotic activity" are used in a
broad sense and refer to the orderly or controlled form of cell
death in mammals that is typically accompanied by one or more
characteristic cell changes, including condensation of cytoplasm,
loss of plasma membrane microvilli, segmentation of the nucleus,
degradation of chromosomal DNA or loss of mitochondrial function.
This activity can be determined and measured, for instance, by cell
viability assays, FACS analysis or DNA electrophoresis, all of
which are known in the art.
[0056] The terms "treating," "treatment," and "therapy" as used
herein refer to curative therapy, prophylactic therapy, and
preventative therapy.
[0057] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell
cancer, small-cell lung cancer, non-small cell lung cancer,
blastoma, gastrointestinal cancer, renal cancer, pancreatic cancer,
glioblastoma, neuroblastoma, cervical cancer, ovarian cancer, liver
cancer, stomach cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial carcinoma, salivary
gland carcinoma, kidney cancer, liver cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma and various types
of head and neck cancer.
[0058] The term "mammal" as used herein refers to any mammal
classified as a mammal, including humans, cows, horses, dogs and
cats.
[0059] The terms "antigen" and "immunogen" are used interchangeably
herein to refer to a molecule or substance which induces an immune
response (preferably an antibody response) in an animal immunized
therewith (i.e. the antigen is "immunogenic" in the animal). The
antigen may be a protein, peptide, carbohydrate, nucleic acid,
lipid, hapten or other naturally occurring or synthetic compound.
Preferably the antigen is a "protein" having a molecular weight of
greater than about 4 kD. The protein may, for example, be a
cellular, bacterial or viral protein.
[0060] By "different antigens" is meant antigens that are
structurally distinct; e.g., in the case of peptides or proteins,
having different amino acid sequences.
[0061] The expression "structurally or functionally related
antigens" refers to antigens with similar structures and/or similar
functions. For example, the antigens may comprise receptors (or
fragments thereof), optionally fused to heterologous amino acid
sequences, which are bound by and/or activated by the same ligand,
e.g., Apo-2L receptors as described herein. Other examples of
structurally and functionally related receptors include members of
the ErbB2 receptor family, such as the EGF receptor, HER2, HER3 and
HER4 receptor; and members of the Rse, Axl and Mer receptor family.
Examples of structurally or functionally related ligands include
the neuregulins, insulin-like growth factors (IGFs), etc.
[0062] The protein antigen of interest may be a "receptor" [i.e. a
protein molecule which exists in nature on a cell surface or within
the cytoplasm of a cell and which is capable of binding to one or
more ligand(s)]. Another exemplary antigen is a protein "ligand"
[i.e. a molecule capable of binding to and, optionally, activating
one or more receptor(s); e.g. a growth factor]. The antigen herein
may, for example, comprise a fragment of a receptor or ligand,
optionally fused to one or more heterologous amino acid sequences
(e.g. the antigen may be an immunoadhesin).
[0063] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the "binding domain" of a
heterologous "adhesin" protein (e.g. a receptor, ligand or enzyme)
with an immunoglobulin constant domain. Structurally, the
immunoadhesins comprise a fusion of the adhesin amino acid sequence
with the desired binding specificity which is other than the
antigen recognition and binding site (antigen combining site) of an
antibody (i.e. is "heterologous") and an immunoglobulin constant
domain sequence. See, e.g., U.S. Pat. No. 5,565,335 and U.S. Pat.
No. 5,116,964, expressly incorporated herein by reference.
[0064] The term "ligand binding domain" as used herein refers to
any native cell-surface receptor or any region or derivative
thereof retaining at least a qualitative ligand binding ability of
a corresponding native receptor. In a specific embodiment, the
receptor is from a cell-surface polypeptide having an extracellular
domain that is homologous to a member of the immunoglobulin
supergenefamily. Other receptors, which are not members of the
immunoglobulin supergenefamily but are nonetheless specifically
covered by this definition, are receptors for cytokines, and in
particular receptors with tyrosine kinase activity (receptor
tyrosine kinases), members of the hematopoietin and nerve growth
factor receptor superfamilies, and cell adhesion molecules, e.g.
(E-, L- and P-) selectins.
[0065] The term "receptor binding domain" is used to designate any
native ligand for a receptor, including cell adhesion molecules, or
any region or derivative of such native ligand retaining at least a
qualitative receptor binding ability of a corresponding native
ligand. This definition, among others, specifically includes
binding sequences from ligands for the above-mentioned
receptors.
[0066] An "antibody-immunoadhesin chimera" comprises a molecule
that combines at least one binding domain of an antibody (as herein
defined) with at least one immunoadhesin (as defined in this
application). Exemplary antibody-immunoadhesin chimeras are the
bispecific CD4-IgG chimeras described in Berg et al., PNAS (USA)
88:4723-4727 (1991) and Chamow et al., J. Immunol. 153:4268
(1994).
[0067] An "isolated" polypeptide is one that has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the polypeptide will be purified (1) to greater than
95% by weight of polypeptide as determined by the Lowry method, and
most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain.
[0068] A "purified" antigen is one which has been subjected to one
or more purification procedures. The purified antigen may be
"homogeneous", which is used herein to refer to a composition
comprising at least about 70% to about 100% by weight of the
antigen of interest, based on total weight of the composition,
preferably at least about 80% to about 100% by weight of the
antigen of interest.
[0069] The term "immunizing" refers to the step or steps of
administering one or more antigens to an animal so that antibodies
can be raised in the animal. Generally, immunizing comprises
injecting the antigen or antigens into the animal. Immunization may
involve one or more administrations of the antigen or antigens.
[0070] The "animal" to be immunized herein is preferably a rodent.
Other animals which can be immunized herein include non-human
primates such as Old World monkey (e.g. baboon or macaque,
including Rhesus monkey and cynomolgus monkey; see U.S. Pat. No.
5,658,570); birds (e.g. chickens); rabbits; goats; sheep; cows;
horses; pigs; donkeys; dogs etc.
[0071] A "rodent" is an animal belonging to the rodentia order of
placental mammals. Exemplary rodents include mice, rats, guinea
pigs, squirrels, hamsters, ferrets etc, with mice being the
preferred rodent for immunizing according to the method herein.
[0072] "Polyclonal antibodies" or "polyclonal antisera" refer to
immune serum containing a mixture of antibodies specific for one
(monovalent or specific antisera) or more (polyvalent antisera)
antigens which may be prepared from the blood of animals immunized
with the antigen or antigens.
[0073] The term "immune cells" refers to cells which are capable of
producing antibodies. The immune cells of particular interest
herein are lymphoid cells derived, e.g. from spleen, peripheral
blood lymphoctes (PBLs), lymph node, inguinal node, Peyers patch,
tonsil, bone marrow, cord blood, pleural effusions and
tumor-infiltrating lymphocytes (TIL).
[0074] By "solid phase" is meant a nonaqueous matrix to which a
molecule of interest can specifically or nonspecifically adhere
(e.g., an assay plate).
[0075] An "adjuvant" is a nonspecific stimulant of the immune
response. The adjuvant may be in the form of a composition
comprising either or both of the following components: (a) a
substance designed to form a deposit protecting the antigen(s) from
rapid catabolism (e.g. mineral oil, alum, aluminium hydroxide,
liposome or surfactant [e.g. pluronic polyol]) and (b) a substance
that nonspecifically stimulates the immune response of the
immunized host animal (e.g. by increasing lymphokine levels
therein). Exemplary molecules for increasing lymphokine levels
include lipopolysaccaride (LPS) or a Lipid A portion thereof;
Bordetalla pertussis; pertussis toxin; Mycobacterium tuberculosis;
and muramyl dipeptide (MDP). Examples of adjuvants include Freund's
adjuvant (optionally comprising killed M. tuberculosis; complete
Freund's adjuvant); aluminium hydroxide adjuvant; and
monophosphoryl Lipid A-synthetic trehalose dicorynomylcolate
(MPL-TDM).
[0076] By "screening" is meant subjecting one or more monoclonal
antibodies (e.g., purified antibody and/or hybridoma culture
supernatant comprising the antibody) to one or more assays which
determine qualitatively and/or quantitatively the ability of an
antibody to bind to an antigen of interest.
[0077] By "immuno-assay" is meant an assay that determines binding
of an antibody to an antigen, wherein either the antibody or
antigen, or both, are optionally adsorbed on a solid phase (i.e.,
an "immunoadsorbent" assay) at some stage of the assay. Exemplary
such assays include ELISAs, radioimmunoassays (RIAs), and FACS
assays.
[0078] An antibody which "cross-reacts" with two or more different
antigens is capable of binding to each of the different antigens,
e.g. as determined by ELISA or FACS as in the examples below.
[0079] An antibody which "specifically cross-reacts" with two or
more different antigens is one which binds to a first antigen and
further binds to a second different antigen, wherein the binding
ability (e.g. OD 450/620; FIGS. 6A-C) of the antibody for the
second antigen at an antibody concentration of about 10 .mu.g/mL is
from about 50% to about 100% (preferably from about 75% to about
100%) of the binding ability of the first antigen as determined in
a capture ELISA as in the examples below. For example, the antibody
may bind specifically to Apo-2 (the "first antigen") and
specifically cross-react with another Apo-2L receptor such as DR4
(the "second antigen"), wherein the extent of binding of about 10
.mu.g/mL of the antibody to DR4 is about 50% to about 100% of the
binding ability of the antibody for Apo-2 in the capture ELISA
herein.
[0080] The word "label" when used herein refers to a detectable
compound or composition which can be conjugated directly or
indirectly to a molecule of interest and may itself be detectable
(e.g., radioisotope labels or fluorescent labels) or, in the case
of an enzymatic label, may catalyze chemical alteration of a
substrate compound or composition which is detectable.
[0081] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the polypeptide nucleic acid.
An isolated nucleic acid molecule is other than in the form or
setting in which it is found in nature. Isolated nucleic acid
molecules therefore are distinguished from the nucleic acid
molecule as it exists in natural cells. However, an isolated
nucleic acid molecule includes a nucleic acid molecule contained in
cells that ordinarily express the polypeptide where, for example,
the nucleic acid molecule is in a chromosomal location different
from that of natural cells.
[0082] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0083] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0084] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0085] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. I.sup.131, I.sup.125, Y.sup.90 and
Re.sup.186) chemotherapeutic agents, and toxins such as
enzymatically active toxins of bacterial, fungal, plant or animal
origin, or variants and/or fragments thereof.
[0086] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include adriamycin, doxorubicin, epirubicin, 5-fluorouracil,
cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa,
busulfan, cytoxin, taxoids, e.g. paclitaxel (TAXOL.TM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.), and doxetaxel
(Taxotere, Rhone-Poulenc Rorer, Antony, Rnace), toxotere,
methotrexate, cisplatin, melphalan, vinblastine, bleomycin,
etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine,
vinorelbine, carboplatin, teniposide, daunomycin, caminomycin,
aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat.
No. 4,675,187), melphalan and other related nitrogen mustards. Also
included in this definition are hormonal agents that act to
regulate or inhibit hormone action on tumors such as tamoxifen and
onapristone.
[0087] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
MODES FOR CARRYING OUT THE INVENTION
A. Mixed Antigen Immunization Protocol
[0088] In one aspect, the invention provides a method for making
monoclonal antibodies wherein an animal is immunized with two or
more different antigens so as to generate polyclonal antibodies,
and preferably monoclonal antibodies, against the two or more
antigens with which the animal was immunized. This method will be
described in more detail in the following sections.
(i) Antigen Selection and Preparation
[0089] The method herein involves preparation of antibodies
directed against one or more different antigens. Preferably, at
least one of the antigens is (and preferably all of the antigens
are) is a biologically important molecule and administration of an
antibody thereagainst to a mammal suffering from a disease or
disorder can result in a therapeutic benefit in that mammal. In the
preferred embodiment of the invention, the antigen is a
protein.
[0090] However, other nonpolypeptide antigens (e.g. tumor
associated glycolipids; see U.S. Pat. No. 5,091,178) may be
used.
[0091] Exemplary protein antigens include molecules such as renin;
a growth hormone, including human growth hormone and bovine growth
hormone; growth hormone releasing factor; parathyroid hormone;
thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating
hormone; calcitonin; luteinizing hormone; glucagon; clotting
factors such as factor VIIIC, factor IX, tissue factor, and von
Willebrands factor; anti-clotting factors such as Protein C; atrial
natriuretic factor; lung surfactant; a plasminogen activator, such
as urokinase or human urine or tissue-type plasminogen activator
(t-PA); bombesin; thrombin; hemopoietic growth factor; tumor
necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated
on activation normally T-cell expressed and secreted); human
macrophage inflammatory protein (MIP-1-alpha); a serum albumin such
as human serum albumin; Muellerian-inhibiting substance; relaxin
A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated
peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a
cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4;
inhibin; activin; vascular endothelial growth factor (VEGF);
receptors for hormones or growth factors; protein A or D;
rheumatoid factors; a neurotrophic factor such as bone-derived
neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3,
NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-.beta.;
platelet-derived growth factor (PDGF); fibroblast growth factor
such as aFGF and bFGF; epidermal growth factor (EGF); transforming
growth factor (TGF) such as TGF-alpha and TGF-beta, including
TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or TGF-.beta.5;
insulin-like growth factor-I and -II (IGF-I and IGF-II);
des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding
proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;
erythropoietin; osteoinductive factors; immunotoxins; a bone
morphogenetic protein (BMP); an interferon such as
interferon-alpha, -beta, and -gamma; colony stimulating factors
(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g.,
IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface
membrane proteins; decay accelerating factor; viral antigen such
as, for example, a portion of the AIDS envelope; transport
proteins; homing receptors; addressins; regulatory proteins;
integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and
VCAM; a tumor associated antigen such as HER2, HER3 or HER4
receptor; and variants and/or fragments of any of the above-listed
polypeptides.
[0092] Preferred molecular targets for antibodies encompassed by
the present invention include CD proteins such as CD3, CD4, CD8,
CD19, CD20 and CD34; members of the ErbB receptor family such as
the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion
molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM and
.alpha.v/.beta.3 integrin including either .alpha. or .beta.
subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b
antibodies); growth factors such as VEGF; IgE; blood group
antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor;
CTLA-4; protein C; an Apo-2L receptor such as Apo-2, DR4, DcR1 and
DcR2; and variants and/or fragments of the above-identified
molecules etc.
[0093] Each antigen to be used in the method is preferably purified
to form an essentially homogeneous preparation of the antigen using
purification techniques available in the art. Examples of
purification procedures which can be used include fractionation on
a hydrophobic interaction chromatography (e.g. on phenyl
sepharose), ethanol precipitation, isoelectric focusing, Reverse
Phase HPLC, chromatography on silica, chromatography on HEPARIN
SEPHAROSE.TM., anion exchange chromatography, cation exchange
chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate
precipitation, hydroxylapatite chromatography, gel electrophoresis,
dialysis, affinity chromatography (e.g. using protein A, protein G,
an antibody, a specific substrate, ligand or antigen as the capture
reagent) or combinations of two or more of these methods.
[0094] In the case of a protein antigen, an immunoadhesin may be
prepared by fusing the protein (or a fragment thereof) to an
immunoglobulin Fc region and purifying the resultant immunoadhesin
by Protein A or Protein G chromatography.
[0095] Soluble antigens or fragments thereof, optionally conjugated
to one or more other molecules, can be used as immunogens for
generating antibodies. For transmembrane molecules, such as
receptors, fragments of these (e.g. the extracellular domain of a
receptor) can be used as the immunogen. Optionally, the protein of
interest or a fragment thereof is fused with a heterologous
molecule, e.g. to form an immunoadhesin as in the examples
below.
[0096] For low molecular weight antigens (such as haptens and
synthetic peptides) and other antigens it may be desirable to
couple the antigen with a "carrier molecule" such as serum albumin
[e.g. bovine serum albumin (BSA)], ovalbumin, keyhole limpet
hemacyanin (KLH), bovine thyroglobulin, soybean trypsin inhibitor
or purified protein derivative of tuberculin (PPD). Such carrier
molecules may be immunogenic in the animal to be immunized (i.e.
they may provide class II-T-cell receptor binding sites). Coupling
may be achieved using a bifunctional coupling agent, such as
maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
carbodiimide, glutaraldehyde, succinic anhydride, SOCl.sub.2, or
R.sup.1N.dbd.C=NR, where R and R.sup.1 are different alkyl groups.
Alternatively, or in addition, the antigen and carrier molecule may
be generated as a fusion protein. In general, approximately 1 mole
of hapten per 50 amino acids of carrier molecule is a reasonable
coupling ratio.
[0097] The antigen may be made more antigenic by coupling to large
matrices, such as agarose beads; chemical coupling to cells (e.g.
red blood cells); converting the antigen to larger compounds by
self-polymerization (e.g. using chemical cross-linkers such as
dinitrophenol or arsynyl, or by partial denaturation); preparing an
immune complex; binding the antigen to nitrocellulose; and/or
binding the antigen to a "carrier" protein (see above).
[0098] In another embodiment, the antigen is present in or on a
cell, bacteria or virus and the host animal is immunized with the
cell, bacteria or virus. Such antigen may be native to the cell,
bacteria or virus or may have been introduced synthetically (e.g.
by recombinant techniques, chemical coupling, etc). Preferably
however, each of the antigens with which the animal is immunized
has been purified by at least one purification step.
(ii) Immunization
[0099] The animal or host to be immunized with the antigens is
selected. In the preferred embodiment, the animal is a rodent, e.g.
a mouse.
[0100] The mouse to be immunized may, for example, be an
"antigen-free" mouse as described in U.S. Pat. No. 5,721,122,
expressly incorporated herein by reference.
[0101] In one embodiment, the host is a transgenic animal in which
human immunoglobulin loci have been introduced. For example, the
transgenic animal may be a mouse comprising introduced human
immunoglobulin genes and one in which the endogenous immunoglobulin
genes have been partially or completely inactivated. Upon
challenge, human antibody production in such transgenic hosts is
observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody
repertoire. This approach is described, for example, in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et
al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg and Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0102] The amount of each antigen administered to the host animal
may, for example, range from about 0.01 .mu.g to about 250 .mu.g,
preferably from about 0.1 .mu.g to about 100 .mu.g. The present
invention involves immunizing the host animal with two or more
different antigens, e.g. from about two to about ten different
antigens, preferably from about three to about four different
antigens. In the preferred embodiment of the invention, the host
animal is immunized with a composition comprising a mixture of the
two or more different antigens and, optionally, a physiologically
acceptable diluent, such as PBS or other buffer. Alternatively, the
animal can be immunized with the antigens separately. The antigens
used to prepare the composition have preferably been purified by at
least by one purification step.
[0103] The host animal may be immunized with the antigens in a
variety of different ways. For example, by subcutaneous,
intramuscular, intradermal, intravenous, and/or intraperitoneal
injections. In addition, injections into lymphoid organs, popliteal
lymph node and/or footpads are possible. It may be desirable to
immunize the animal using a combination of two or more different
administration routes, separately and/or simultaneously.
[0104] Where the primary response is weak, it may be desirable to
boost the animal at spaced intervals until the antibody titer
increases or plateaus. After immunization, samples of serum (test
bleeds) may be taken to check the production of specific
antibodies. Preferably, the host animal is given a final boost
about 3-5 days prior to isolation of immune cells from the host
animal.
(iii) Monoclonal Antibody Production
[0105] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975). In the
hybridoma method, "immune cells" that produce or are capable of
producing polyclonal antibodies are obtained from the animal
immunized as described above. Various immune cells are described
above, with lymph nodes or spleen being the preferred source of
immune cells for generating monoclonal antibodies. Such cells may
then be fused with myeloma cells using a suitable "fusing agent",
such as polyethylene glycol or Sendai virus, to form a hybridoma
cell [Goding, Monoclonal Antibodies: Principles and Practice, pp.
59-103 (Academic Press, 1986)].
[0106] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0107] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and P3X63AgU.1, SP-2 or X63-Ag8-653 cells
available from the American Type Culture Collection, Manassas, Va.,
USA. The 210-RCY3.Ag1.2.3 rat myeloma cell line is also available.
Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the production of human monoclonal antibodies
[Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)].
[0108] Alternatively, hybridoma cell lines may be prepared from the
immune cells of the immunized animal in other ways, e.g. by
immortalizing the immune cells with a virus (e.g. with Epstein Barr
Virus), or with an oncogene in order to produce an immortalized
cell line producing the monoclonal antibody of interest. See, also,
Babcook et al. PNAS (USA), 93:7843-7848 (1996), concerning
production of monoclonal antibodies by cloning immunoglobulin cDNAs
from single cells producing specific antibodies for yet another
strategy for preparing monoclonal antibodies using immune cells of
the immunized animal.
(iv) Screening
[0109] Screening is performed to identify one or more monoclonal
antibodies capable of binding to each antigen. Generally, one
screen for antibodies which bind to each antigen with which the
animal has been immunized. Such screening may be performed on
culture supernatant and/or purified antibodies, from each hybridoma
culture supernatant resulting from fusion. Alternatively, or in
addition, screening may be carried out using culture supernatant
and/or purified antibodies from cloned hybridoma cells (see below).
In addition, where cross-reactive antibodies are of interest, the
ability of the monoclonal antibodies to cross-react with two or
more different antigens may be determined. Moreover, it may be
desirable to screen for antibodies with certain functional
characteristics (e.g. agonistic activity, blocking activity,
etc).
[0110] The binding specificity of monoclonal antibodies produced by
hybridoma cells may, for example, be determined in an immuno-assay,
e.g. by immunoprecipitation or other in vitro binding assay, such
as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay
(ELISA).
[0111] There are three general classes of screening methods that
can be employed (a) antibody capture assays; (b) antigen capture
assays; and (c) functional screens.
[0112] In antibody capture assays, the antigen may be bound to a
solid phase, monoclonal antibodies to be tested are allowed to bind
to the antigen, unbound antibodies are removed by washing, and then
the bound antibodies are detected, e.g. by a secondary reagent such
as a labeled antibody that specifically recognizes the
antibody.
[0113] For an antigen capture assay, the antigen may be labeled
directly (various labels are described herein). In one embodiment,
monoclonal antibodies to be tested may be bound to a solid phase
and then reacted with the optionally labeled antigen.
Alternatively, the antibody-antigen complex may be allowed to form
by immunoprecipitation prior to binding of the monoclonal antibody
to be tested to a solid phase. Once the antibody-antigen complexes
are bound to the solid phase, unbound antigen may be removed by
washing and positives may be identified by detecting the
antigen.
[0114] Various functional screens exist for identifying monoclonal
antibodies with desired activities. Examples include the agonistic
activity assay and blocking assay of the examples below;
keratinocyte monolayer adhesion assay and the mixed lymphocyte
response (MLR) assay [Werther et al. J. Immunol. 157:4986-4995
(1996)]; tumor cell growth inhibition assays (as described in WO
89/06692, for example); antibody-dependent cellular cytotoxicity
(ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. Pat.
No. 5,500,362); and hematopoiesis assays (see WO 95/27062). The
class/subclass of the antibodies may be determined, e.g., by
double-diffusion assays; antibody capture on antigen-coated plates;
and/or antibody capture on anti-IgG antibodies.
[0115] To screen for antibodies which bind to a particular epitope
on the antigen of interest (e.g., those which block binding of any
of the antibodies disclosed herein to an
[0116] Apo-2L receptor), a routine cross-blocking assay such as
that described in Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. Alternatively, epitope mapping, e.g. as described in
Champe et al., J. Biol. Chem. 270:1388-1394 (1995), can be
performed to determine whether the antibody binds an epitope of
interest.
[0117] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, single-cell
clones may be subcloned by limiting dilution procedures [Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)]; single cell cloning by picks; or cloning
by growth in soft agar [Harlow and Lane, Antibodies: A Laboratory
Manual Cold Spring Harbor Laboratory (1988); pps 224-227].
[0118] Hybridoma clones may be grown by standard methods. Suitable
culture media for this purpose include, for example, DMEM or
RPMI-1640 medium. In addition, the hybridoma cells may be grown in
vivo as ascites tumors in an animal. [Harlow and Lane, Antibodies:
A Laboratory Manual Cold Spring Harbor Laboratory (1988); Chapter
7].
[0119] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein G or A-Sepharose, hydroxylapatite chromatography,
gel electrophoresis, dialysis, or affinity chromatography.
(v) Cloning and Further Modifications of the MAb
[0120] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Recombinant production of antibodies will be described
in more detail below.
[0121] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences [U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851
(1984)], or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0122] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0123] In one embodiment, the monoclonal antibody is humanized.
Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof [such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies] which contain minimal sequence derived
from non-human immunoglobulin. A humanized antibody has one or more
amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0124] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody [Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)]. Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies [Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993)].
[0125] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0126] The antibodies of the invention may also be prepared as
monovalent antibodies. Methods for preparing monovalent antibodies
are well known in the art. For example, one method involves
recombinant expression of immunoglobulin light chain and modified
heavy chain. The heavy chain is truncated generally at any point in
the Fc region so as to prevent heavy chain crosslinking.
Alternatively, the relevant cysteine residues are substituted with
another amino acid residue or are deleted so as to prevent
crosslinking.
[0127] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
Papain digestion of antibodies typically produces two identical
antigen binding fragments, called Fab fragments, each with a single
antigen binding site, and a residual Fc fragment. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen combining
sites and is still capable of cross-linking antigen.
[0128] The Fab fragments produced in the antibody digestion also
contain the constant domains of the light chain and the first
constant domain (CH.sub.1) of the heavy chain. Fab' fragments
differ from Fab fragments by the addition of a few residues at the
carboxy terminus of the heavy chain CH.sub.1 domain including one
or more cysteines from the antibody hinge region. Fab'-SH is the
designation herein for Fab' in which the cysteine residue(s) of the
constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0129] It may be desirable to generate a multispecific antibody
comprising the monoclonal antibody. Multispecific antibodies have
binding specificities for at least two different antigens. While
such molecules normally will only bind two antigen (i.e. bispecific
antibodies, BsAbs), antibodies with additional specificities such
as trispecific antibodies are encompassed by this expression when
used herein. Bispecific antibodies can be prepared as full length
antibodies or antibody fragments [e.g. F(ab').sub.2 bispecific
antibodies].
[0130] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
[Millstein et al., Nature, 305:537-539 (1983)]. Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0131] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0132] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this symmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0133] According to another approach described in WO96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the C.sub.H3 domain of an antibody constant domain.
In this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0134] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0135] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0136] The invention also pertains to immunoconjugates comprising
the antibody described herein conjugated to a cytotoxic agent such
as a chemotherapeutic agent, toxin (e.g. an enzymatically active
toxin of bacterial, fungal, plant or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0137] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof which can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugate
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y and .sup.186Re.
[0138] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al. Science, 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody., See WO94/11026.
[0139] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide).
[0140] Immunoliposomes comprising the antibody may also be
prepared. Liposomes containing the antibody are prepared by methods
known in the art, such as described in Epstein et al., Proc. Natl.
Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad.
Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and
4,544,545. Liposomes with enhanced circulation time are disclosed
in U.S. Pat. No. 5,013,556.
[0141] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al. J.
National Cancer Inst. 81(19):1484 (1989)
[0142] The antibody of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0143] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form.
[0144] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful, for converting
non-toxic 5-fluorocytosine into the anti-cancer drug,
5-fluorouracil; proteases, such as serratia protease, thermolysin,
subtilisin, carboxypeptidases and cathepsins (such as cathepsins B
and L), that are useful for converting peptide-containing prodrugs
into free drugs; D-alanylcarboxypeptidases, useful for converting
prodrugs that contain D-amino acid substituents;
carbohydrate-cleaving enzymes such as .beta.-galactosidase and
neuraminidase useful for converting glycosylated prodrugs into free
drugs; .beta.-lactamase useful for converting drugs derivatized
with .beta.-lactams into free drugs; and penicillin amidases, such
as penicillin V amidase or penicillin G amidase, useful for
converting drugs derivatized at their amine nitrogens with
phenoxyacetyl or phenylacetyl groups, respectively, into free
drugs. Alternatively, antibodies with enzymatic activity, also
known in the art as "abzymes", can be used to convert the prodrugs
of the invention into free active drugs [see, e.g., Massey, Nature
328: 457-458 (1987)]. Antibody-abzyme conjugates can be prepared as
described herein for delivery of the abzyme to a tumor cell
population.
[0145] The enzymes of this invention can be covalently bound to the
antibody by techniques well known in the art such as the use of the
heterobifunctional crosslinking reagents discussed above.
Alternatively, fusion proteins comprising at least the
antigen-binding region of an antibody of the invention linked to at
least a functionally active portion of an enzyme of the invention
can be constructed using recombinant DNA techniques well known in
the art [see, e.g., Neuberger et al., Nature, 312: 604-608
(1984)].
[0146] In certain embodiments of the invention, it may be desirable
to use an antibody fragment, rather than an intact antibody, to
increase tumor penetration, for example. In this case, it may be
desirable to modify the antibody fragment in order to increase its
serum half life. This may be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment (e.g. by mutation of the appropriate region in
the antibody fragment or by incorporating the epitope into a
peptide tag that is then fused to the antibody fragment at either
end or in the middle, e.g., by DNA or peptide synthesis).
[0147] The salvage receptor binding epitope preferably constitutes
a region wherein any one or more amino acid residues from one or
two loops of a Fc domain are transferred to an analogous position
of the antibody fragment. Even more preferably, three or more
residues from one or two loops of the Fc domain are transferred.
Still more preferred, the epitope is taken from the CH2 domain of
the Fc region (e.g., of an IgG) and transferred to the CH1, CH3, or
V.sub.H region, or more than one such region, of the antibody.
Alternatively, the epitope is taken from the CH2 domain of the Fc
region and transferred to the C.sub.L region or V.sub.L region, or
both, of the antibody fragment. See, e.g., U.S. Pat. No. 5,747,035,
expressly incorporated herein by reference.
[0148] Covalent modifications of the antibody are included within
the scope of this invention. They may be made by chemical synthesis
or by enzymatic or chemical cleavage of the antibody, if
applicable. Other types of covalent modifications of the antibody
are introduced into the molecule by reacting targeted amino acid
residues of the antibody with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues.
[0149] The antibodies may optionally be covalently attached or
conjugated to one or more chemical groups. A polyol, for example,
can be conjugated to an antibody molecule at one or more amino acid
residues, including lysine residues as disclosed in WO 93/00109.
Optionally, the polyol is a poly(alkelene glycol), such as
poly(ethylene glycol) (PEG), however, those skilled in the art
recognize that other polyols, such as, for example, poly(propylene
glycol) and polyethylene-polypropylene glycol copolymers, can be
employed using techniques for conjugating PEG to polypeptides. A
variety of methods for pegylating polypeptides have been described.
See, e.g. U.S. Pat. No. 4,179,337 which discloses the conjugation
of a number of hormones and enzymes to PEG and polypropylene glycol
to produce physiologically active compositions having reduced
immunogenicities.
[0150] The antibodies may also be fused or linked to another
heterologous polypeptide or amino acid sequence such as an epitope
tag.
B. Anti-Apo-2L Receptor Antibodies
[0151] The present also provides antibodies which are able to
cross-react with two or more different Apo-2L receptors. These
cross-reactive antibodies may be prepared according to the mixed
antigen immunization method described above (or by immunizing an
animal with a single antigen, e.g. Apo-2 or another Apo-2L
receptor), or may be made by other techniques such as those
elaborated below.
[0152] As described in the Examples below, anti-Apo-2 monoclonal
antibodies have been prepared. Three of these antibodies
(3H1.18.10, 3H3.14.5 and 3D5.1.10) have been deposited with the
ATCC. In one embodiment, the monoclonal antibodies of the invention
will have the same biological characteristics as one or more of the
monoclonal antibodies secreted by the three hybridoma cell lines
deposited with the ATCC producing antibodies 3H1.18.10, 3H3.14.5 or
3D5.1.10. The term "biological characteristics" is used to refer to
the in vitro and/or in vivo activities or properties of the
monoclonal antibody, such as the ability to specifically bind to
Apo-2 and/or another Apo-2L receptor, or to substantially block,
induce or enhance Apo-2L receptor activation. Optionally, the
monoclonal antibody will bind to the same epitope as one or more of
the 3H1.18.10, 3H3.14.5 or 3D5.1.10 antibodies disclosed herein.
The monoclonal antibody preferably has the hypervariable region
residues of one or more of the above-mentioned antibodies, e.g., it
may comprise a humanized variant.
[0153] Aside from the methods described above for obtaining
antibodies (by immunizing a host with one or more antigens), other
techniques are available for generating anti-Apo-2L receptor
antibodies. For example, human antibodies can be produced in phage
display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381
(1992); Marks et al., J. Mol. Biol., 222:581 (1991)]. The
techniques of Cole et al. and Boerner et al. are also available for
the preparation of human monoclonal antibodies [Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].
Suitable methods for preparing phage libraries have been reviewed
and are described in Winter et al., Annu. Rev. Immunol., 12:433-55
(1994); Soderlind at al., Immunological Reviews, 130:109-123
(1992); Hoogenboom, Tibtech February 1997, Vol. 15; Neri et al.,
Cell Biophysics, 27:47-61 (1995). Libraries of single chain
antibodies may also be prepared by the methods described in WO
92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO
95/01438 and WO 95/15388. Antibody libraries are also commercially
available, for example, from Cambridge Antibody Technologies
(C.A.T.), Cambridge, UK.
C. Recombinant Antibodies
[0154] The invention also provides isolated nucleic acid encoding
an antibody as disclosed herein (e.g. as obtained by mixed antigen
immunization and/or an anti-Apo-2L receptor antibody), vectors and
host cells comprising the nucleic acid, and recombinant techniques
for the production of such antibodies.
[0155] For recombinant production of the antibody, the nucleic acid
encoding it is isolated and inserted into a replicable vector for
further cloning (amplification of the DNA) or for expression. DNA
encoding the monoclonal antibody is readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding
the heavy and light chains of the antibody). Many vectors are
available. The vector components generally include, but are not
limited to, one or more of the following: a signal sequence, an
origin of replication, one or more marker genes, an enhancer
element, a promoter, and a transcription termination sequence.
Examples of such expression system components are disclosed in U.S.
Pat. No. 5,739,277 issued Apr. 14, 1998, expressly incorporated
herein by reference.
[0156] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
(see, e.g., U.S. Pat. No. 5,739,277, expressly incorporated herein
by reference.)
[0157] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0158] The host cells used to produce the antibody of this
invention may be cultured in a variety of media. Any necessary
supplements may also be included at appropriate concentrations that
would be known to those skilled in the art. The culture conditions,
such as temperature, pH, and the like, are those previously used
with the host cell selected for expression, and will be apparent to
the ordinarily skilled artisan.
[0159] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Where the antibody is secreted
into the medium, supernatants from such expression systems are
generally first concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. A protease inhibitor such as PMSF may be
included in any of the foregoing steps to inhibit proteolysis and
antibiotics may be included to prevent the growth of adventitious
contaminants.
[0160] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
D. Therapeutic Uses for Antibodies
[0161] The antibodies described herein have therapeutic utility.
Agonistic Apo-2L receptor antibodies, for instance, may be employed
to activate or stimulate apoptosis in cancer cells. Accordingly,
the invention provides methods for treating cancer using
antibodies, such as cross-reactive Apo-2L receptor antibodies. It
is of course contemplated that the methods of the invention can be
employed in combination with still other therapeutic techniques
such as surgery.
[0162] The antibody is preferably administered to the mammal in a
carrier. Suitable carriers and their formulations are described in
Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack
Publishing Co., edited by Oslo et al. Typically, an appropriate
amount of a pharmaceutically-acceptable salt is used in the
formulation to render the formulation isotonic. Examples of a
pharmaceutically-acceptable carrier include saline, Ringer's
solution and dextrose solution. The pH of the solution is
preferably from about 5 to about 8, and more preferably from about
7 to about 7.5. Further carriers include sustained release
preparations such as semipermeable matrices of solid hydrophobic
polymers containing the antibody, which matrices are in the form of
shaped articles, e.g., films, liposomes or microparticles. It will
be apparent to those persons skilled in the art that certain
carriers may be more preferable depending upon, for instance, the
route of administration and concentration of antibody being
administered.
[0163] The antibody can be administered to the mammal by injection
(e.g., intravenous, intraperitoneal, subcutaneous, intramuscular),
or by other methods such as infusion that ensure its delivery to
the bloodstream in an effective form. The antibody may also be
administered by intratumoral, peritumoral, intralesional, or
perilesional routes, to exert local as well as systemic therapeutic
effects. Local or intravenous injection is preferred.
[0164] Effective dosages and schedules for administering the
antibody may be determined empirically, and making such
determinations is within the skill in the art. Those skilled in the
art will understand that the dosage of antibody that must be
administered will vary depending on, for example, the mammal which
will receive the antibody, the route of administration, the
particular type of antibody used and other drugs being administered
to the mammal. Guidance in selecting appropriate doses for antibody
is found in the literature on therapeutic uses of antibodies, e.g.,
Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges
Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357;
Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et
al., eds., Raven Press, New York (1977) pp. 365-389. A typical
daily dosage of the antibody used alone might range from about 1
.mu.g/kg to up to 100 mg/kg of body weight or more per day,
depending on the factors mentioned above.
[0165] The antibody may also be administered to the mammal in
combination with effective amounts of one or more other therapeutic
agents or in conjunction with radiation treatment. Therapeutic
agents contemplated include chemotherapeutics as well as
immunoadjuvants and cytokines. The antibody may be administered
sequentially or concurrently with the one or more other therapeutic
agents. The amounts of antibody and therapeutic agent depend, for
example, on what type of drugs are used, the cancer being treated,
and the scheduling and routes of administration but would generally
be less than if each were used individually.
[0166] Following administration of antibody to the mammal, the
mammal's cancer and physiological condition can be monitored in
various ways well known to the skilled practitioner. For instance,
tumor mass may be observed physically or by standard x-ray imaging
techniques.
[0167] The Apo-2L receptor antibodies of the invention may also be
useful in enhancing immune-mediated cell death in cells expressing
Apo-2L receptor(s), for instance, through complement fixation or
ADCC. Alternatively, antagonistic anti-Apo-2L receptor antibodies
may be used to block excessive apoptosis (for instance in
neurodegenerative disease) or to block potential
autoimmune/inflammatory effects of Apo-2 resulting from NF-KB
activation. Such antagonistic antibodies can be utilized according
to the therapeutic methods and techniques described above.
E. Non-therapeutic Uses for Antibodies
[0168] Antibodies may further be used in diagnostic assays for
their antigen, e.g., detecting its expression in specific cells,
tissues, or serum. Various diagnostic assay techniques known in the
art may be used, such as competitive binding assays, direct or
indirect sandwich assays and immunoprecipitation assays conducted
in either heterogeneous or homogeneous phases [Zola, Monoclonal
Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp.
147-158]. The antibodies used in the diagnostic assays can be
labeled with a detectable moiety. The detectable moiety should be
capable of producing, either directly or indirectly, a detectable
signal. For example, the detectable moiety may be a radioisotope,
such as .sup.3H, .sup.14C, .sup.32P, .sup.35S, or .sup.125I, a
fluorescent or chemiluminescent compound, such as fluorescein
isothiocyanate, rhodamine, or luciferin, or an enzyme, such as
alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
Any method known in the art for conjugating the antibody to the
detectable moiety may be employed, including those methods
described by Hunter et al., Nature, 144:945 (1962); David et al.,
Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,
40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407
(1982).
[0169] Antibodies also are useful for the affinity purification of
antigen from recombinant cell culture or natural sources. In this
process, the antibodies are immobilized on a suitable support, such
as Sephadex resin or filter paper, using methods well known in the
art. The immobilized antibody then is contacted with a sample
containing the antigen to be purified, and thereafter the support
is washed with a suitable solvent that will remove substantially
all the material in the sample except the antigen, which is bound
to the immobilized antibody. Finally, the support is washed with
another suitable solvent that will release the antigen from the
antibody.
F. Kits Containing Antibodies
[0170] In a further embodiment of the invention, there are provided
articles of manufacture and kits containing antibodies which can be
used, for instance, for the therapeutic or non-therapeutic
applications described above. The article of manufacture comprises
a container with a label. Suitable containers include, for example,
bottles, vials, and test tubes. The containers may be formed from a
variety of materials such as glass or plastic. The container holds
a composition which includes an active agent that is effective for
therapeutic or non-therapeutic applications, such as described
above. The active agent in the composition is the antibody, e.g. an
Apo-2L receptor antibody. The label on the container indicates that
the composition is used for a specific therapy or non-therapeutic
application, and may also indicate directions for either in vivo or
in vitro use, such as those described above.
[0171] The kit of the invention will typically comprise the
container described above and one or more other containers
comprising materials desirable from a commercial and user
standpoint, including buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0172] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0173] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
Example 1
Preparation of Immunogens
[0174] The receptor antigens in Examples 2 and 3 below were all
receptors for Apo-2 ligand [Pitti et al., J. Biol. Chem.,
271:12687-12690 (1996); and WO97/25428]. The Apo-2L receptors were:
DR4 [Pan et al., Science, 276:111-113 (1997)]; Apo-2 [called "DR5"
in Sheridan et al., Science 277:818-821 (1997)]; DcR1 [Sheridan et
al., Science 277:818-821 (1997)]; and DcR2 [Marsters et al., Curr.
Biol., 7:1003-1006 (1997)].
[0175] Receptor immunoadhesins (designated "DR4-IgG", "Apo-2-IgG",
"DcR1-IgG" and "DcR2-IgG") were prepared by fusing the
extracellular domain sequence of each receptor to the hinge and Fc
region of human immunoglobulin G.sub.1 heavy chain in pRK5 as
described previously [Ashkenazi et al., Proc. Natl. Acad. Sci.,
88:10535-10539 (1991)]. The immunoadhesin proteins were expressed
by transient transfection into human 293 cells and purified from
cell supernatants by protein A affinity chromatography, as
described by Ashkenazi et al., supra. Purified immunoadhesin was
suspended in phosphate buffered saline (PBS).
Example 2
Mixed Antigen Immunization
[0176] Animals in this example were immunized with the four
receptor immunoadhesins of the preceding example. The mixed antigen
immunization scheme used is shown in FIG. 1.
[0177] Balb/c mice (obtained from Charles River Laboratories) were
immunized into each hind foot pad 14 times at 3-4 day intervals,
with a mixture of DR4-IgG, Apo-2-IgG, DcR1-IgG and DcR2-IgG (1
.mu.g each) suspended in monophosphoryl lipid A plus trehalose
dicorynomycolate adjuvant (MPL-TDM; Ribi Immunochem. Research Inc.,
Hamilton, Mont.) at a 1:1 ratio of immunoadhesin:adjuvant (DcR2-IgG
was only included in the mixture used for the final six
immunizations).
[0178] Three days after the final boost, popliteal lymph node cells
nodes were removed from the mice and a single cell suspension was
prepared in DMEM media (obtained from Biowhitakker Corp.)
supplemented with 1% penicillin-streptomycin. The lymph node cells
were fused with murine myeloma cells P3X63AgU.1 (ATCC CRL 1597)
using 35% polyethylene glycol and cultured in 96-well culture
plates.
[0179] Hybridomas were selected in super DMEM [DMEM plus 10% fetal
calf serum (FCS), 10% NCTC-109 (BioWittaker, Wakersville, Md.), 100
mM pyruvate, 100 U/ml insulin, 100 mM oxaloacetic acid, 2 mM
glutamine, 1% nonessential amino acids (GIBCO), 100 U/ml penicillin
and 100 .mu.g/ml streptomycin] containing 100 .mu.M hypoxanthine,
0.4 .mu.M aminopterin, and 16 .mu.M thymidine (1.times.HAT, Sigma
Chemical Co., St. Louis, Mo.).
[0180] Ten days after the fusion, 180 .mu.l of each hybridoma
culture supernatant was screened for the presence of antibodies to
three different antigens (i.e. DR4-IgG, Apo-2-IgG and CD4-IgG
control) in a capture ELISA. Hybridoma cells were re-fed with 200
.mu.l of super DMEM containing 10% FCS and antibiotics. Two days
later, 180 .mu.l of culture supernatant was collected and screened
for the presence of antibodies to another two different antigens
(i.e. DcR1-IgG and DcR2-IgG) in a capture ELISA. After careful
examination of the ELISA results, potential positive hybridomas
secreting monoclonal antibodies against each antigen were cloned
twice using a limiting dilution method. Culture supernatants from
these clones were re-tested for their ability to bind to a
particular antigen, but not to others, including CD4-IgG, in a
capture ELISA. Isotypes of the antibodies were also determined.
[0181] Selected clones were also tested for (a) their ability to
recognize Apo-2L receptors expressed on cell membranes by flow
cytometry (FACS); (b) their ability to block the ligand-receptor
interaction, and (c) for their agonistic activity.
Example 3
Single Antigen Immunization
[0182] The single antigen immunization scheme is shown in FIG. 2.
The general method was almost the same as the mixed antigen
immunization protocol in Example 2 above, except that only a single
antigen was used as the immunogen and during the screening of
hybridomas supernatant (180 .mu.l) was collected only once to
screen for the presence of positive monoclonal antibodies to the
particular antigen and control CD4-IgG.
[0183] Balb/c mice (obtained from Charles River Laboratories) were
immunized by injecting 0.5 .mu.g/50 .mu.l of immunoadhesin protein
(diluted in MPL-TDM adjuvant purchased from Ribi Immunochemical
Research Inc., Hamilton, Mont.) 10 times into each hind foot pad at
3-4 day intervals. Three days after the final boost, popliteal
lymph nodes were removed from the mice and a single cell suspension
was prepared in DMEM media (obtained from Biowhitakker Corp.)
supplemented with 1% penicillin-streptomycin. The lymph node cells
were then fused with murine myeloma cells P3X63AgU.1 (ATCC CRL
1597) using 35% polyethylene glycol and cultured in 96-well culture
plates. Hybridomas resulting from the fusion were selected in HAT
medium as in Example 2. Ten days after the fusion, hybridoma
culture supernatants (180 .mu.l) were screened in an ELISA to test
for the presence of monoclonal antibodies binding to the
immunoadhesin protein.
Example 4
Capture ELISA
[0184] For the capture ELISA, 96-well microtiter plates (Maxisorb;
Nunc, Kamstrup, Denmark) were coated by adding 50 .mu.l of 2
.mu.g/ml goat anti-human IgG Fc (purchased from Cappel
Laboratories) in PBS to each well and incubating at 4.degree. C.
overnight. The plates were then washed three times with wash buffer
(PBS containing 0.05% TWEEN 20.TM.). The wells in the microtiter
plates were then blocked with 50 .mu.l of 2.0% bovine serum albumin
(BSA) in PBS and incubated at room temperature for 1 hour. The
plates were, then washed again three times with wash buffer.
[0185] After the washing step, 50 .mu.l of 1 .mu.g/ml immunoadhesin
protein (as described above) in assay buffer (PBS plus 0.5% BSA)
was added to each well. The plates were incubated for 1 hour at
room temperature on a shaker apparatus, followed by washing three
times with wash buffer.
[0186] Following the wash steps, 100 .mu.l of the hybridoma
supernatants or purified antibody (using Protein G-sepharose
columns) (1 .mu.g/ml) was added to designated wells. 100 .mu.l of
P3X63AgU.1 myeloma cell conditioned medium was added to other
designated wells as controls. The plates were incubated at room
temperature for 1 hour on a shaker apparatus and then washed three
times with wash buffer.
[0187] Next, 50 .mu.l HRP-conjugated goat anti-mouse IgG Fc
(purchased from Cappel Laboratories), diluted 1:1000 in assay
buffer (0.5% bovine serum albumin, 0.05% % TWEEN 20.TM., 0.01%
Thimersol in PBS), was added to each well and the plates incubated
for 1 hour at room temperature on a shaker apparatus. The plates
were washed three times with wash buffer, followed by addition of
50 .mu.l of substrate (TMB microwell peroxidase substrate,
Kirkegaard & Perry, Gaithersburg, Md.) to each well and
incubation at room temperature for 10 minutes. The reaction was
stopped by adding 50 .mu.l of TMB 1-component stop solution
(diethyl glycol, Kirkegaard & Perry) to each well, and
absorbance at 450 nm was read in an automated microtiter plate
reader.
Example 5
Antibody Isotyping
[0188] The isotypes of antibodies were determined by coating
microtiter plates with isotype specific goat anti-mouse Ig (Fisher
Biotech, Pittsburgh, Pa.) overnight at 4.degree. C. The plates were
then washed with wash buffer. The wells in the microtiter plates
were then blocked with 200 .mu.l of 2% bovine serum albumin and
incubated at room temperature for one hour. The plates were washed
again three times with wash buffer. Next, 100 .mu.l of hybridoma
culture supernatant or 5 .mu.g/ml of purified antibody was added to
designated wells. The plates were incubated at room temperature for
30 minutes and then 50 HRP-conjugated goat anti-mouse IgG (as
described above) was added to each well. The plates were incubated
for 30 minutes at room temperature. The level of HRP bound to the
plate was detected using HRP substrate as described above.
Example 6
Flow Cytometry
[0189] FACS analysis was performed using 9D cells (a human B
lymphoid cell line expressing Apo-2 and DR4; Genentech, Inc.) or
human microvascular endothelial (HUMEC) cells (Cell Systems, Inc.),
expressing DcR1 and DcR2.
[0190] Twenty-five microliters of cell suspension (4.times.10.sup.6
cells/ml) in cell sorter buffer (PBS containing 1% FCS and 0.02%
NaN.sub.3) was added to U-bottom microtiter wells, mixed with 100
.mu.l of culture supernatant or purified monoclonal antibody
(purified on Protein-G sepharose column) (10 .mu.g/ml) in cell
sorter buffer (CSB), and incubated for 30 min on ice. After
washing, cells were incubated with 100 .mu.l of FITC-conjugated
goat anti-mouse IgG for 30 min at 4.degree. C. Cells were washed
twice in CSB and resuspended in 150 .mu.l of CSB and analyzed by
FACScan (Becton Dickinson, Mountain View, Calif.).
Example 7
Assay for Antibody Ability to Block Apo-2L-Induced Apoptosis
[0191] Hybridoma supernatants and purified antibodies were tested
for their ability to block Apo-2 ligand induced 9D cell apoptosis.
Human 9D cells (5.times.10.sup.5 cells) were suspended in 50 .mu.l
of complete RPMI medium (RPMI plus 10% FCS, glutamine, nonessential
amino acid, penicillin and streptomycin and sodium pyrubate) in
Falcon 2052 tubes. 10 .mu.g of antibody plus 10 .mu.g of DR4
antibody in 200 .mu.l of medium was added to cells and cells were
incubated on ice for 15 minutes. 0.5 .mu.g of Apo-2L (soluble
His-tagged Apo-2L prepared as described in WO 97/25428; see also
Pitti et al., supra) in 250 .mu.l of complete RPMI was added to
cells. 9D cells were incubated overnight at 37.degree. C. in the
presence of 7% CO.sub.2. Cells were harvested and washed once in
PBS. The viability of the cells was then determined by the staining
of FITC-Annexin V binding to phosphatidylserine according to
manufacturer's recommendations (Clontech). Briefly cells washed in
PBS were resuspended in 200 .mu.l of binding buffer. Ten .mu.l of
Annexin V-FITC (1 .mu.g/ml) and 10 .mu.l of propidium iodide were
added to the cells. After incubation for 15 min in the dark, cells
were analyzed by FACScan.
Example 8
Apoptosis by Monoclonal Antibodies after Crosslinking with
Anti-Mouse Ig
[0192] Human 9D cells (2.5.times.10.sup.5 cells) in 50 .mu.l of
complete RPMI medium (RPMI plus 10% FCS, glutamine, nonessential
amino acid, penicillin and streptomycin and sodium pyruvate) were
added to Falcon 2052 tubes. Cells were then incubated with 10 .mu.g
of monoclonal antibody in 100 .mu.l of complete RPMI medium on ice
for 15 min. Cells were then incubated with 10 .mu.g of goat
anti-mouse IgG Fc in 350 .mu.l of complete RPMI medium overnight at
37.degree. C. After washing once with PBS, cells were resuspended
in 200 .mu.l of PBS containing 0.5% BSA and incubated with 10 .mu.l
of FITC-Annexin and 10 .mu.l of propidium iodide for 15 min in the
dark. Dead cells then detected by FACScan as described above.
Results and Discussion
[0193] FIGS. 3 and 4 provide a comparison of the antigen specific
sera titer from mice immunized with a single antigen (FIG. 3)
verses mice immunized with mixed antigens (FIG. 4).
[0194] Sera titers (EC50) from mice immunized with each antigen
were approximately 10,000 for each specific antigen. Antigen
specific sera titers (EC50) of mice immunized with mixed antigens
were .about.10,000 for DR4-IgG, Apo-2-IgG, DcR1-IgG and
.about.5,000 for DcR2-IgG. Accordingly, the antigen specific
antibody titers were quite comparable whether mice were immunized
with individual antigen or with a mixture of four different
antigens. The DcR2-IgG specific titer (1:4,000) of mice immunized
with four different antigens was slightly lower than that
(1:10,000) of mice immunized with DcR2-IgG alone. However, this may
have been due to the fact that the mice immunized with mixed
antigens received DcR2-IgG only 6 times, while mice immunized with
DcR2-IgG alone received 10 injections.
TABLE-US-00001 TABLE 1 COMPARISON BETWEEN SINGLE ANTIGEN AND MIXED
ANTIGEN IMMUNIZATIONS DR4 Apo-2 DcR2 Single Mixed Single Mixed
Single Mixed Antigen Antigen Antigen Antigen Antigen Antigen ELISA
Positive 13.30% 6.50% 4.50% 2.10% 1.20% 2.30% FACS Positive 48%
.sup. 17% .sup. 36% .sup. 46% .sup. 20% 0 Final mono- 5 3 4 5 1 0
clonal antibody selected Specificity 3/5 1/3 1/4 1/5 Cross- 0/5 1/3
0/4 1/5 Reactive* *Specifically cross-react with both DR4 and
Apo-2
[0195] Table 1 compares the effectiveness of generating monoclonal
antibodies to DR4, Apo-2 and DcR2 using mice immunized with a
single antigen, verses mice immunized with mixed antigens. One can
generate monoclonal antibodies using both methods. However, the
mixed antigen immunization scheme resulted in the production and
isolation of more antibodies that cross-reacted with different
receptors (i.e., recognized shared epitopes between two proteins;
see Table 1). In particular, using the mixed antigen immunization
protocol, antibodies were identified which cross-reacted with
different Apo-2L receptors. The cross-reactivities as determined by
capture ELISA are shown in Table 2.
TABLE-US-00002 TABLE 2 ANTIBODY CROSS-REACTIVITIES WITH APO-2L
RECEPTORS Cross Reactivity Isotype DR4 Apo-2 DcR1 DcR2 3H1.18.10 G1
+/- +++ +/- +/- 3H3.14.5 G1 +/- +++ +/- +/- 3D5.1.10 G1 ++ +++ -
+/- ++ .gtoreq.75% binding (compared to Apo-2 binding) + ~50-74%
binding +/- ~25-49% binding - .ltoreq.24% binding
[0196] As shown in FIG. 6C and Table 2, the 3D5.1.10 antibody
specifically bound Apo-2 and specifically cross-reacted with DR4.
Antibodies 3H1.18.10 and 3H3.14.5 specifically bound Apo-2 and
displayed some cross-reactivity with other Apo-2L receptors tested.
(Table 2 and FIGS. 6A and 6B) Other biological activities of the
antibodies from Table 2 were evaluated according to the methods
described in Example 6 (antibody binding to cell-surface receptor);
Example 7 (blocking or neutralizing ability); and Example 8
(apoptotic activity). The results are shown in Table 3 below.
TABLE-US-00003 TABLE 3 OTHER ACTIVITIES OF THE ANTI-APO-2L RECEPTOR
ANTIBODIES FACS of Blocking Apoptotic 9D cells ability activity
3H1.18.10 + - - 3H3.14.5 + + + 3D5.1.10 + - -
[0197] All three antibodies were able to bind Apo-2 expressed on
the surface of 9D cells. The 3H3.14.5 antibody was also able to
inhibit apoptosis induced via interaction between Apo-2L and Apo-2.
This antibody was further capable of inducing apoptosis of 9D cells
in the presence of an anti-Fc antibody to cross-link
antibodies.
Deposit of Material
[0198] The following materials have been deposited with the
American Type Culture Collection, 10801 University Blvd., Manassas,
Va., USA (ATCC):
TABLE-US-00004 Material ATCC Dep. No. Deposit Date pRK5-Apo-2
209021 May 8, 1997 3F11.39.7 HB-12456 Jan. 13, 1998 3H1.18.10
HB-12535 Jun. 2, 1998 3H3.14.5 HB-12534 Jun. 2, 1998 3D5.1.10
HB-12536 Jun. 2, 1998
[0199] This deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposit will be made available by ATCC under the terms
of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC Section 122 and the
Commissioner's rules pursuant thereto (including 37 CFR Section
1.14 with particular reference to 8860G 638).
[0200] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0201] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
211799DNAhuman 1cccacgcgtc cgcataaatc agcacgcggc cggagaaccc
cgcaatctct 50gcgcccacaa aatacaccga cgatgcccga tctactttaa gggctgaaac
100ccacgggcct gagagactat aagagcgttc cctaccgcca tggaacaacg
150gggacagaac gccccggccg cttcgggggc ccggaaaagg cacggcccag
200gacccaggga ggcgcgggga gccaggcctg ggctccgggt ccccaagacc
250cttgtgctcg ttgtcgccgc ggtcctgctg ttggtctcag ctgagtctgc
300tctgatcacc caacaagacc tagctcccca gcagagagcg gccccacaac
350aaaagaggtc cagcccctca gagggattgt gtccacctgg acaccatatc
400tcagaagacg gtagagattg catctcctgc aaatatggac aggactatag
450cactcactgg aatgacctcc ttttctgctt gcgctgcacc aggtgtgatt
500caggtgaagt ggagctaagt ccctgcacca cgaccagaaa cacagtgtgt
550cagtgcgaag aaggcacctt ccgggaagaa gattctcctg agatgtgccg
600gaagtgccgc acagggtgtc ccagagggat ggtcaaggtc ggtgattgta
650caccctggag tgacatcgaa tgtgtccaca aagaatcagg catcatcata
700ggagtcacag ttgcagccgt agtcttgatt gtggctgtgt ttgtttgcaa
750gtctttactg tggaagaaag tccttcctta cctgaaaggc atctgctcag
800gtggtggtgg ggaccctgag cgtgtggaca gaagctcaca acgacctggg
850gctgaggaca atgtcctcaa tgagatcgtg agtatcttgc agcccaccca
900ggtccctgag caggaaatgg aagtccagga gccagcagag ccaacaggtg
950tcaacatgtt gtcccccggg gagtcagagc atctgctgga accggcagaa
1000gctgaaaggt ctcagaggag gaggctgctg gttccagcaa atgaaggtga
1050tcccactgag actctgagac agtgcttcga tgactttgca gacttggtgc
1100cctttgactc ctgggagccg ctcatgagga agttgggcct catggacaat
1150gagataaagg tggctaaagc tgaggcagcg ggccacaggg acaccttgta
1200cacgatgctg ataaagtggg tcaacaaaac cgggcgagat gcctctgtcc
1250acaccctgct ggatgccttg gagacgctgg gagagagact tgccaagcag
1300aagattgagg accacttgtt gagctctgga aagttcatgt atctagaagg
1350taatgcagac tctgccwtgt cctaagtgtg attctcttca ggaagtgaga
1400ccttccctgg tttacctttt ttctggaaaa agcccaactg gactccagtc
1450agtaggaaag tgccacaatt gtcacatgac cggtactgga agaaactctc
1500ccatccaaca tcacccagtg gatggaacat cctgtaactt ttcactgcac
1550ttggcattat ttttataagc tgaatgtgat aataaggaca ctatggaaat
1600gtctggatca ttccgtttgt gcgtactttg agatttggtt tgggatgtca
1650ttgttttcac agcacttttt tatcctaatg taaatgcttt atttatttat
1700ttgggctaca ttgtaagatc catctacaaa aaaaaaaaaa aaaaaaaaag
1750ggcggccgcg actctagagt cgacctgcag aagcttggcc gccatggcc
17992411PRThumanxaa410xaa = leu or met 2Met Glu Gln Arg Gly Gln Asn
Ala Pro Ala Ala Ser Gly Ala Arg 1 5 10 15Lys Arg His Gly Pro Gly
Pro Arg Glu Ala Arg Gly Ala Arg Pro 20 25 30Gly Leu Arg Val Pro Lys
Thr Leu Val Leu Val Val Ala Ala Val 35 40 45Leu Leu Leu Val Ser Ala
Glu Ser Ala Leu Ile Thr Gln Gln Asp 50 55 60Leu Ala Pro Gln Gln Arg
Ala Ala Pro Gln Gln Lys Arg Ser Ser 65 70 75Pro Ser Glu Gly Leu Cys
Pro Pro Gly His His Ile Ser Glu Asp 80 85 90Gly Arg Asp Cys Ile Ser
Cys Lys Tyr Gly Gln Asp Tyr Ser Thr 95 100 105His Trp Asn Asp Leu
Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp 110 115 120Ser Gly Glu Val
Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr 125 130 135Val Cys Gln
Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro 140 145 150Glu Met
Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val 155 160 165Lys
Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His 170 175
180Lys Glu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val 185
190 195Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys
200 205 210Val Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly
Asp 215 220 225Pro Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala
Glu Asp 230 235 240Asn Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro
Thr Gln Val 245 250 255Pro Glu Gln Glu Met Glu Val Gln Glu Pro Ala
Glu Pro Thr Gly 260 265 270Val Asn Met Leu Ser Pro Gly Glu Ser Glu
His Leu Leu Glu Pro 275 280 285Ala Glu Ala Glu Arg Ser Gln Arg Arg
Arg Leu Leu Val Pro Ala 290 295 300Asn Glu Gly Asp Pro Thr Glu Thr
Leu Arg Gln Cys Phe Asp Asp 305 310 315Phe Ala Asp Leu Val Pro Phe
Asp Ser Trp Glu Pro Leu Met Arg 320 325 330Lys Leu Gly Leu Met Asp
Asn Glu Ile Lys Val Ala Lys Ala Glu 335 340 345Ala Ala Gly His Arg
Asp Thr Leu Tyr Thr Met Leu Ile Lys Trp 350 355 360Val Asn Lys Thr
Gly Arg Asp Ala Ser Val His Thr Leu Leu Asp 365 370 375Ala Leu Glu
Thr Leu Gly Glu Arg Leu Ala Lys Gln Lys Ile Glu 380 385 390Asp His
Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu Gly Asn 395 400 405Ala
Asp Ser Ala Xaa Ser 410
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